Certain proteins require the presence of sulfhydryl groups in their structure for activity and stability. In the presence of oxidizing agents, however, including atmospheric oxygen, the sulfhydryl groups are oxidized to disulfide linkages with concomitant deleterious changes in the protein's properties. These problems are magnified when the protein is in serum-based material or lyophilized.
Addition of excess thiols has been utilized as a means of protection for the sulfhydryl groups of proteins. A large number of thiols has been evaluated for effectiveness as a protective agent against oxidation (G. Szasz, "Proceedings of the Second International Symposium on Clinical Enzymology", 1975, pages III-1 to 36). Szasz examined twenty-seven thiol compounds with creatine kinase, an unstable enzyme. Both possible dimercapto (1,3 and 1,2-dimercapto) glycerols were tested for their effectiveness but monothioglycerol [1-mercapto (or 1-sulfhydryl)-2,3-propanediol] was not included in the evaluation.
Monothioglycerol (MTG, 1-sulfhydryl-2,3-propanediol) has been found to be effective for this purpose; its half-life in solutions in equilibrium with atmospheric oxygen is sufficiently long for practical purposes. However, it has been stated by L. G. Morin, in Clinical Chemistry, Volume 23, No. 9, 1569 (1977), at page 1574 that "(monothioglycerol) would undoubtedly not lyophilize". For this reason, MTG has not been utilized as a sulfhydryl-protector in lyophilized materials.
A. P. MacKenzie, at the International Symposium on Freeze-Drying of Biological Products, Washington, D.C., October, 1976, Proceedings, page 52, reported the lyophilisis of glycerol-containing biological materials. In absence of residual moisture content determinations, however, the artisan is not guided as to the lyophilizability of glycerol-containing materials. Moreover, even if glycerol-containing materials are lyophilizable, the artisan again is not guided as to the class of lyophilizable low molecular weight liquids.
G. Szasz, et al. [Clinical Chemistry, Volume 24, No. 9, 1557 (1978)] also investigated MTG but observed gelling of the serum proteins, with 50 mM MTG per liter, after six hours storage at 37.degree. C. These investigators then selected N-acetyl cystein as the protecting agent of choice for creatine kinase isoenzymes.